Western blot

Wet blot was run with a nitrocellulose membrane (Hybond-ECL) in ice cold towin buffer for 2 h at 300 mA. Afterwards, membrane was washed in TNT buffer for 5 min and incubated in blocking solution for 1 h. After cutting off the marker lane from the membrane, the residual membrane was incubated for 1 h with the first antibody (diluted in an adequate concentration in blocking solution). Following three washing processes for 10 min in TNT, the membrane was incubated with a second antibody (HRP-conjugated) in blocking solution, while the biotinylated marker membrane was incubated in streptavidin-conjugated antibody (1:5000) in TNT buffer for 1 h. The membrane was then again washed three times in TNT, incubated for 1 min in ECL solution (mixture of solution A + solution B, ratio 1:1) and chemiluminescence was detected after appropriate exposure times from 5 s up to 15 min with a CCD camera using a FujiLAS 1000 imaging station.

3.2.14 Immunofluorescence

3.2.14.1 Analysis of hPARP-1 from adherent cells

All adherent cells were seeded on sterile glass coverslips (12 mm) at a density of 2x10⁴ cells/cm² in a 12-well culture dish and were allowed to adhere overnight. Cells were then washed once with PBS (2 ml) and fixed with 4% paraformaldehyde (1 ml) for 20 min at RT.

Subsequently, the supernatant was removed and cells were incubated with glycine solution for 2 min. After supernatant removal, cells were permeabilised for 5 min with 0.4% triton X-100 in PBS following three washings with PBS. Cells were then incubated in a humid chamber (custom made) with monoclonal antibody FI-23 directed against hPARP-1 (dilution of 1:250 in blocking solution) at 37°C for 1 h. After three washings with PBS, antibody-antigen complexes were detected with Alexa Fluor488-conjugated goat anti-mouse secondary antibody at 37°C for 45 min. The cells were washed three times, counterstained with Hoechst 33342, washed four times, mounted on a glass slide and were finally examined under a fluorescence microscope for detection of hPARP-1 protein.

3.2.14.2 Analysis of PAR in suspension cells

To analyze PAR formation in EL-4 cells after X-irradiation, cells were grown to 5x10⁵ cells/ml, centrifuged at 200 g for 5 min and were then resuspended and counted. Cell number was adjusted to 1x10⁶cells/ml and aliquoted to 100 µl/Eppendorf tube (2 ml). After addition of PARP inhibitors (PJ34, BYK204165 or BYK236864), cells were incubated at 37°C for 30 min. Cells were then X-irradiated on ice at 40 Gy. After that PAR formation could take place for 3 min in a water bath at 37°C. Thereafter, cells were kept on ice to prevent PAR degradation by PARG. Cells were then transferred quickly in the cone-shaped borehole of a cytospin container and centrifuged at 200 g for 5 min at 0°C to stick them to the glass slide underneath. Following this, the cells were then air dried with nitrogen gas and completely dried by exposing them at 37°C for 15 min. Cells were then fixed with pre-cooled (-20°C) methanol/acetic acid (3:1, v/v) for 10 min on ice. The following steps were performed in the same way as in section “analysis of PAR in fibroblasts”.

3.2.14.3 Analysis of PAR in fibroblasts

3T3 cells from Parp-1^+/+ and Parp-1^-/- mice were grown and passaged in 3T3 cell culture medium to confluence. Then, the cells were washed in PBS, trypsinised and plated on sterile

coverslips (12 mm) at a density of 2x10⁴ cells/cm² in 12-well culture dishes, and were allowed to adhere overnight (Wagner et al., 2007). After exposure of the cultures to PARP inhibitors (BYK204165 or BYK236864; 0.3 – 10 µM, final DMSO concentration 0.3%) for 30 min, cells were first washed with PBS and after that PAR formation was stimulated by treatment with H2O2 (5 mM for Parp-1^+/+ fibroblasts, 50 mM for Parp-1^-/- fibroblasts) for 5 min at 37°C. The cells were then fixed with methanol/acetic acid (3:1, v/v) for 10 min at RT. After three washings with PBS, cells were incubated with monoclonal antibody 10H directed against PAR at a dilution of 1:250 in blocking solution for 1 h at 37°C in a humid chamber. After three washings with PBS, antibody-antigen complexes were detected with Alexa Fluor488-conjugated goat anti-mouse secondary antibody for 45 min at 37°C. The cells were washed three times and counterstained with DAPI. Coverslips were mounted on glass slides and examined under a fluorescence microscope for detection of PAR.

3.2.14.4 Cytotoxicity assay (microscopy)

After DNA damage in COR4 and COMF10 cells by alkylating agents (MNNG, MMS), applied in the absence or presence of dexamethasone (Dex), apoptosis, necrosis and cell viability were measured by microscopy. COR4 cells (Meyer et al., 2000) are stable transfectants expressing human glucocorticoid receptor derived from the parental SV40 transformed Chinese ovary hamster cells (Kupper et al., 1995) and served as a control cell line. COMF10 cells have in addition to COR4 cell line a plasmid (pPARP93), encoding the full length human PARP-1 cDNA under the transcriptional control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, and pTKneo (Meyer et al., 2000).

The MMTV promoter is induced by glucocorticoid hormone (here: Dex) via the glucocorticoid receptor, which then leads to hPARP-1 expression in the COMF10 cells, but not in the COR4 cells, due to the missing pPARP93 plasmid. Cells were grown for two days in a cell culture flask to 80% confluence without selection antibiotics, washed in PBS, trypsinised, centrifuged (200 g, 5 min, RT) and finally resuspended in a small volume of DMEM medium. Cells were counted and adjusted to 2x10⁴ cells/ml in stimulation medium containing Dex (100 nM) or not. Then 100 µl/well (2000 cells/well) were transferred into a flat bottom 96-well plate. After 24 h cells were treated with N-methyl-N´-nitro-N-nitrosoguanidine (MNNG, 2.5 - 20 µM, in triplicate) or methyl methanesulfonate (MMS, 100 - 750 µM, in triplicate) diluted in the corresponding medium (+/- Dex), and were incubated at 37°C for 24 h in a CO₂ incubator.

Each well was then treated with 5 µl pre-diluted (1:10 in PBS) Sytox^®/Hoechst mix for 5 min.

The number of necrotic (Sytox^® stained), apoptotic (i.e. condensed/fragmented nuclei, Hoechst stained) and healthy cells (i.e. round nuclei, Hoechst stained) were scored with a fluorescence microscope. A total number of 1,800 cells were assessed for each experimental condition and cell type by examining 200 cells/well in three independent experiments performed in triplicate (Eltze and Kunzmann et al., submitted).